Calcif Tissue Int (1991) 49:421-426
Calcified Tissue International 9 1991 Springer-Verlag New York Inc.
Molecular and Cellular Biology Expression of Bone Sialoprotein (BSP) in Developing Human Tissues Paolo Bianco, Larry W. Fisher, Marian F. Young, John D. Termine, and Pamela Gehron Robey Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Building 30, Room 106 Bethesda, Maryland 20892, USA Received September 24, 1990, and in revised form December 3, 1990.
Summary. Bone sialoprotein (BSP) and its messenger RNA were localized in developing human skeletal and nonskeletal tissues by means of immunohistochemistry and in situ hybridization. Both protein and mRNA were found in mature, bone-forming cells but not in their immature precursors. In addition, osteoclasts displayed positive immunostaining and high densities of autoradiographic grains by in situ hybridization experiments. BSP was expressed in fetal epiphyseal cartilage cells, particularly in hypertrophic chondrocytes of growth plates. Though neither the protein nor the mRNA were identified in a variety of other connective and nonconnective tissues, an unexpected finding was the expression of BSP in the trophoblast cells of placenta. These findings show that BSP is primarily an osteoblast-derived component of the bone matrix expressed at late stages of differentiation. We have also found that osteoclasts produce BSP, possibly as a mediator of cell attachment to bone. Key words: Bone sialoprotein - - Developing bone - Noncollagenous proteins - - In situ hybridization - - Osteoblast - - Osteoclast - - Cell adhesion.
A sialoprotein of Mr 25,000 was first purified from bovine bone by Herring et al. [I]. Fisher et al. [2] later showed that this 25,000 Mr molecule was most likely a degradation product of a larger -70,000 M r molecule (predicted M r -33,000 by cDNA sequence analysis [3]) and was known briefly as BSP II. Bone sialoprotein (BSP) is found in a variety of mammals, including rat, human, and rabbit [4, 5]. Interestingly, in the rabbit, BSP exists as a keratan sulfate proteoglycan [6]. Both rat [7] and human [3] BSP have recently been cloned and sequenced and have been localized to human chromosome 4 [3]. BSP contains an Arg-Gly-Asp (RGD) sequence, which represents the recognition site for integrins, the cell membrane receptors in a variety of extracellular matrix proteins involved in cell adhesion [8]. In fact, BSP was used to identify an RGD-directed vitronectin-type receptor in rat osteosarcoma cells [9], and mediates fibroblast [10] as well as bone cell [11] attachment in vitro. Evidence that BSP is an endogenous product of normal bone cells comes from in vitro studies with bovine and murine bone-derived cells [2, 12]. Expression in transformed bone Offprint requests to: P. Gehron Robey
cell lines is variable. UMR 106, and in particular UMR-106NIH, produce large amounts of BSP constituitively [13] whereas ROS 17/2.8 produce BSP only after induction [7]. This raises the issue of whether certain subsets (or maturational stages) of bone cells, possibly represented by clonal cell lines, may be specific sites of BSP production in intact tissue. Finally, previous data based on chemical extractions have indicated a highly specific expression of BSP in bone [3, 14], but no immunolocalization studies that document the precise localization or tissue distribution in bone or nonskeletal tissue have been reported to date. In this study we report the expression and localization of BSP in developing human tissues as revealed by immunostaining and in situ hybridization.
Material and Methods Tissues
Human tissues of gestational age 14-17 weeks obtained from therapeutic procedures were placed immediately in Dulbecco's Minimal Essential Medium (DMEM), and dissected within 2 hours. Ribs, calvaria, long bones of the limbs, placentae, and a variety of tissues and viscera (ocular tissue, tendon, skin, skeletal muscle, kidney, sclera, lung) were dissected and fixed for 2 hours or overnight in 4% formaldehyde (freshly made from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.2 and embedded in paraffin. Bone samples were decalcified (0.3 M ethylene diamine tetraacetic acid, 0.15 M NaCI, 0.01 M Tris-HC1 pH 7.2) in buffered EDTA before embedding (2--4 hours), in paraffin (melting point 58-60~ and 5 ~m sections were prepared. Prior to use, slides bearing sections were heated on a warming tray (60~) just until the paraffin melted, and placed in sequential washes of 100% xylene, 100% ethanol, and 70% ethanol (5 minutes in each wash). Antiserum to B S P
A rabbit antiserum (LF 6) raised against purified human BSP was used in this study. Details on the production of the antiserum were given elsewhere [5]. This antibody recognizes only BSP in Western blotting procedures and does not cross-react with osteopontin. Because BAG-75, another bone Sialoprotein isolated from rat bone, has not yet been identified in human bone, it is unknown whether LF-6 cross-reacts with this protein, but it is unlikely based on the immunoblotting procedures and blocking experiments described below. Immunostaining
An indirect immunoperoxidase protocol was used. Deparaffinized sections mounted on Poly-L-lysine-coated slides were exposed to
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Fig. 2. Immunolocalization of BSP in osteoclasts. (A) An osteoclast adherent to the tip of a primary trabecula. Arrows point to a bandlike zone of immunostaining at the cell/bone interface. (B) A tangentially cut osteoclast, viewed "en face," shows multiple cytoplasmic loci of labeling. Arrows point to "rings" of reaction product around individual nuclei. Counterstain with hemalum. Bar = 20 ~m.
Preparation of RNA Probe
Fig. 1. Immunolocalization of BSP in bone-forming cells. Subperiosteal bone formation in human fetal rib. (A) Labeling of individual osteoid-forming osteoblasts (arrows). Note the presence of a single roundish paranuclear dot of staining (C, arrow). (B) Positive staining of young osteocytes embedded in newly formed bone matrix. Bar = 25 ~m (A), 10 p.m (B), 6 ~m (C). The pattern of cytoplasmic staining in an individual osteoblast is shown in C.
The template for transcription of 35S-labeled RNA probes was a pBluescript plasmid (B6-5g) containing a 1165 base pair insert with the complete protein-encoding sequence of human BSP [3]. Both antisense and sense (control) 35S-UTP-labeled probes (specificity activity 2 x 108 cpm/p~g) were synthesized using the Riboprobe Gemini System (Promega). To synthesize the antisense probe, the plasmid was linearized with BamHI and transcribed using T7 RNA polymerase. To synthesize the sense (control) probe, the plasmid was linearized with Kpn I and transcribed with T3 RNA polymerase. After removal of the template with DNAse RQ 1, the probes were purified by centrifugation on a Sephadex G-50 'spin' column (Pharmacia), and reduced in size by limited alkaline hydrolysis [16].
In Situ
0.3% H202 to inhibit endogenous peroxidase, washed, and exposed to normal goat serum (1:5 in phosphate-buffered saline, PBS, 0.02 M phosphate 0.15 M sodium chloride pH 7.4) for 20 minutes. The antibody to BSP was used at a dilution of 1:40 in PBS, 0.1% bovine serum albumin (BSA). Incubation lasted for 2 hours at room temperature. An affinity purified, peroxidase-labeled goat anti-rabbit IgG antibody was used as the second antibody at a dilution of 1:30 (with an incubation time of 45' at room temperature). Peroxidase activity was revealed using the diamino benzidine reaction [15]. Controls included substitution of normal rabbit serum for the primary antibody, and an antigen absorption test. Blocking experiments were carried out by incubating 1 nmol of purified human BSP with 100 ~1 of the working dilution of the BSP antibody overnight at 4'C, followed by centrifugation at 10,000 x g and subsequent use in immunostaining. The BSP antibody was also preincubated with purified human bone osteopontin (BSP I) to show specificity for BSP and the lack of cross-reactivity in the BSP antiserum with human bone osteopontin.
Chondroitinase Digestion In some experiments, sections were digested with Chondroitin ABC lyase (protease-free, from Proteus Vulgaris, ICN Biochemicals) before immunostaining. The enzyme was used at a concentration of 1.25 U/ml in 0.1 M Tris, 0.05 M calcium acetate, 0.1% BSA, pH 7.2, and incubation lasted for 10 minutes at 37~
Hybridization
Deparaffinized sections were treated with 0.2 N HCI, digested with proteinase K (from Tritirachium album, Sigma), 1 ixg/ml in 10 mM Tris, 2 mM CaC12 for 15 minutes at 37~ washed in PBS, and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0. The sections were washed in PBS, then in 2 x SSC, dehydrated in ascending concentrations of ethanol, and air dried. The hybridization mixture consisted of 50% formamide, 0.3 M NaC1, 20 mM Tris HC1, pH 8.0, 1 mM EDTA, 1 • Denhardt's solution, tRNA 500 ixg/ml, 10 mM DTT, 10% dextran sulfate, and 2 • 105 cpm/ixl of probe. The volume of hybridization solution added was varied according to the dimensions of the section, and the sections were covered with small plastic squares cut out of heat-sealable, Seal-a-Meal TM pouches. The slides were hybridized overnight at 52~ in a humid atmosphere. The next day, the plastic squares were removed by immersing the slides in 50% formamide, 0.3 M NaC1, 20 mM Tris HC1, pH 8.0, 1 mM EDTA, 1 • Denhardt's, 10 mM DTT. The slides were then sequentially washed in 50% formamide, 4 • SSC, 10 mM DTT at 52~ for 30 minutes, 50% formamide, 2 x SSC, 10 mM DTT at 52~ for 30 minutes; 2 x SSC (4 • 5 minutes) at room temperature. Adventitiously bound probe was removed by digesting the sections with 20 ~g/ml RNase A and 1 i~g/ml RNase T1 in 0.5 M NaC1, 10 mM Tris HC1, pH 8.0, 1 mM EDTA, for 30 minutes at 37~ The slides were then washed in RNase buffer, then in 2 • SSC, 10 mM DTT for 30 minutes at room temperature; 0.1 • SSC, 10 mM DTT at 52~ for 15 minutes; 2 • SSC for 10 minutes at room temperature; then dehydrated and air dried. For autoradiography, the slides were dipped in a 1:1 dilution of Kodak NTB-2 emulsion, allowed to rest vertically at room temperature for 5 hours, then exposed at 4~ for 3-8 days in the presence of desiccant. Exposed
P. Bianco et al.: Bone BSP in Developing Tissues
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Fig. 3. Immunolocalization of BSP in placenta. Overview (A) and detail of placental villi (B). Arrows point to areas of surface labeling of the villar profile. Hemalum counterstain. Bar = 40 I~m (A), 25 ixm (B).
slides were developed in Kodak D-19 developer for 2.5 minutes at 15~ stopped in 1% acetic acid, fixed in Kodak fixer, washed in running tap water, counterstained with hemalum, dehydrated in ethanol, cleared in xylene, and mounted in Permount. The slides were viewed in both brightfield and darkfield microscopy.
Results
Immunolocalization of BSP In developing bone, BSP was specifically localized to boneforming cells, i.e., mature osteoblasts, and young osteocytes (osteoid osteocytes) recently entombed in deposited bone matrix (Fig. 1A, B). The staining pattern displayed by bone cells was distinctive in that it consisted of a discrete, sharp, and roundish perinuclear dot, the size, location, and shape of which were consistent with staining of the Golgi apparatus (Fig. 1C). Not all osteoblasts on bone surfaces were positively stained and could reflect different biosynthetic activities of certain cells, or the random orientation of the plane of sectioning which would not necessarily run through the presumed Golgi apparatus of all cells. Preosteogenic ceils in the inner layer of the periostium remained unstained, as did the fibroblastic cells in the outer layer. Osteoclasts also displayed positive immunostaining (Fig. 2). The reaction product in these cells was distributed at the cell/bone interface, and/or as multiple perinculear rings and cytoplasmic dots. Staining of bone matrix was intense but uneven, with focal linear features occasionally reminiscent of cement lines. Focal granular staining was seen in osteoid at the borderline with mature mineralized matrix. Hypertrophic chondrocytes in the growth plate, especially those located at their lateral aspects (i.e., facing the bony collar), consistently showed positive cytoplasmic staining. Occasional cytoplasmic staining of randomly distributed resting chondrocytes was also observed in epiphyseal cartilage. Cartilage matrix remained completely unstained in undigested sections, but was faintly positive in sections predigested with Chondroitin ABC lyase. None of the other connective and nonconnective tissues investigated (ocular tissues, skin, tendon, muscle, kidney etc.) showed any significant staining for BSP. A notable exception was represented by placental villi, where immuno-
staining was seen associated with the surface of ceils of both the syncytial trophoblast and cytotrophoblast (Fig. 3). Foci of " s p o t t y " placental calcification and the endothelial lining of occasional capillaries in the core of villi were also stained. No staining was seen in control sections incubated with either normal rabbit serum of BSP-absorbed antiserum. The immunostaining was completely blocked by preincubation of the antibody with highly purified human bone sialoprotein indicating the specificity of the antibody and the lack of cross-reactivity with other proteins such as osteopontin and the potential human analog of BAG-75. In addition, positive staining was not affected by absorption of the antiserum with osteopontin.
Localization of BSP mRNA In developing subperiosteal bone, BSP mRNA was localized to mature osteoblasts on bone surfaces and young osteocytes within bone matrix (Fig. 4A-D), but not in the preosteogenic and fibrous layers of the periosteum. Autoradiographic grains were observed over Howship's lacunae (Fig. 4E-F), which suggested that osteoclasts were also labeled. The morphology of the labeled cells at those sites was somewhat obscured by the high density of the grains themselves. Individual osteoclasts that had become attached from bone (most likely by the sectioning procedure) were easier to identify in our sections on the basis of their size and multinuclearity. Such cells also displayed high hybridization signal (Fig. 4G, H). In nonbone tissues, positive hybridization signals were detected only in epiphyseal cartilage and placenta. In epiphyseal cartilage, relatively low levels of BSP m R N A (as judged by grain densities) were observed in resting and proliferating chondrocytes, and higher levels in hypertrophic chondrocytes, indicating a gradient of increasing expression of BSP mRNA in the growth plate (Fig. 5A, B). In placenta, cells of cyto- and syncytial trophoblast at the surface of villi displayed strong hybridization signals (Fig. 6). No positive labeling was seen over any of the other tissues investigated (skin, kidney, eye, muscle, etc.) and in sections hybridized with the sense probe. The distribution of BSP in matrix and cells, along with localization of BSP mRNA, is summarized in Table 1.
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P. Bianco et al.: Bone BSP in Developing Tissues
Fig. 4. Localization of BSP mRNA in bone. Brightfield and darkfield images of the bony collar of human developing femur at low power (A, B, bar = 50 ~m) and high power (C, D). (Bar = 20 Ixm). Osteoblasts at the bone surface (large arrows) and osteocytes within the matrix (small arrows) are labeled. Bar = 50 0.m. Brightfield and darkfield images of trabeculae of primary spongiosa (E, F). Note the concentration of autoradiographic grains over a Howship's lacuna (arrow). Bar = 50 gin. Detail of a labeled osteoclast lying in proximity of a bone trabecula (G, It). Nomarski optics is used in (G) to give enough contrast to the bone trabecula (bt). Autoradiographic grains are shown in (H), where arrows point to individual nuclei. Bar = 30 p~m.
Discussion
The present study conclusively demonstrates that in developing human bone, osteoblasts and young osteocytes are the primary source of bone sialoprotein. The peculiar intracellular staining pattern displayed by bone-forming cells with our anti-BSP antibody is strongly reminiscent of the one described by Mark et al. [17] for osteopontin in rat bone cells, and shown to result from localization of osteopontin in the Golgi apparatus [18]. This would be consistent with the known occurrence of siaiylation in the Golgi and the high content of sialic acid of both proteins, which would tend to cause accumulation in this organelle. Interestingly, not all osteoblasts and young osteocytes were stained, the ramifications of which are not clear, but could represent either subpopulations of cells or differences in the plane of section. It is of interest that only mature bone-forming cells (which include osteoblasts sitting at bone surfaces and young
osteocytes) express BSP detectable by these methods. The level o f BSP expression was undetectable in less mature cells, such as preosteoblasts in the inner periostium, compared with the more differentiated cells. This " l a t e " expression in bone-forming cells is different from that of other noncollagenous components of bone matrix, such as osteonectin [19] and the small proteoglycans biglycan (PG I) and decorin (PG II) which are expressed earlier in the differentiation pathway of osteoblasts [20], but similar to that of osteopontin [17] with the exception that small mononuclear cells in the marrow were osteopontin positive, but not BSP positive at this early stage of development. The programmed expression of specific matrix components by bone-forming cells at different maturational stages raises the possibility that distinct biosynthetic profiles mark specific phenotypes within the bone cell lineage. An intriguing finding of this study is the localization of BSP protein and of the corresponding m R N A in osteoclasts.
P. Bianco et al.: Bone BSP in Developing Tissues
425
Fig. 5. Detection of BSP mRNA in epiphyseal cartilage. Note the higher grain density over hypertrophic chondrocytes (bottom) as compared with proliferating cells (top). Brightfield (A) and darkfield (B) images. Bar = 50 ~m.
Fig. 6. Detection of BSP mRNA in placenta. Brightfield (A) and darkfield (B) images of a placental villus showing strong hybridization signal over trophoblastic cells (arrows). Bar = 60 txm. Though the localization of the protein in osteoclasts is clear in terms of cell morphology, localization of matrix proteins within osteoclasts may be due to either diffusion artifacts or localization of molecules in the process of being resorbed rather than synthesized. Nevertheless, (1) antibodies to other matrix proteins used on the same materials did not yield any staining of osteoclasts; (2) a discrete cytoplasmic staining pattern, consisting of multiple perinuclear rings, was observed in osteoclasts, which might be consistent with a Golgi staining, given the "ring-like" perinuclear distribution of the Golgi apparatuses in osteoclasts [21]; (3) the observation of positive signals, indicative of mRNA, over resorption lacunae and individual osteoclasts in our in situ hybridization experiments supports the notion that osteoclasts can synthesize BSP. Given the postulated role of the BSP in mediating cell attachment, the possibility that osteoclasts may use endogenously produced cell adhesion molecules to attach to bone surfaces is most interesting. Preliminary results indicate that isolated chicken osteoclasts do indeed adhere to BSP in vitro (F. P. Ross, personal communication).
Our results show that although restricted to a limited number of cell types, BSP expression is not totally unique to bone cells. The expression of BSP mRNA in epiphyseal cartilage predominantly in hypertrophic chondrocytes, and the observed gradient of increasing expression along the direction of chondrocyte maturation in growth plates are reminiscent of similar reported localizations of other bone-enriched noncollagenous proteins and/or of the c o r r e s p o n d i n g mRNAs, such as osteonectin [14, 19, 22] and osteopontin [18]. It is worth noting here that the expression of high levels of BSP mRNA might be an additional example of the appearance or up-regulation of certain biosynthetic or phenotypic features characteristic of mature osteoblasts, including alkaline phosphatase activity, in hypertrophic chondrocytes of growth plates. In addition to the hard tissue localization, trophoblasts are the only nonskeletal source of BSP at 14 weeks of gestation. Thus, for trophoblasts, phenotypic modulations cannot account for what could be called an "ectopic" expression of a bone-enriched protein. The known occurrence of
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Table 1. Distribution of BSP and BSP mRNA in human developing skeletal and nonskeletal tissues
Bone Matrix Preosteoblasts Mature osteoblasts Osteocytes Osteoclasts Cartilage Matrix Chondrocytes Resting Proliferating Hypertrophic Nonskeletal tissues Soft tissues Parenchymal viscera Trophoblast
BSP
BSP mRNA
+ + + +
+ + +
-+a • • +
• +
+
+
BSP = bone sialoprotein; + = positive; - = negative; • = variably positive in some cells a Weakly positive only after ABCase
d y s t r o p h i c m i n e r a l i z a t i o n (as o p p o s e d to m a t r i x - m e d i a t e d m i n e r a l i z a t i o n ) in p l a c e n t a , a n d t h e i m m u n o l o c a l i z a t i o n of B S P in s u c h p l a c e n t a l s p o t t y foci o f m i n e r a l d e p o s i t i o n , also does n o t p r o v i d e a s a t i s f a c t o r y a n a l o g y with skeletal tissues. However, the association of BSP and mineral may not be fortuitous. It is i n t e r e s t i n g to n o t e t h a t b o t h o s t e o c l a s t s a n d t r o p h o b l a s t cells are i n v o l v e d in t h e f o r m a t i o n of s y n c y t i a , for w h i c h cell-cell a d h e s i o n is n e c e s s a r y , a n d t h a t t h e adhesion b e t w e e n fetal a n d m a t e r n a l tissues is o n e o f the m o s t intriguing a n d l e a s t u n d e r s t o o d p h e n o m e n a of g e s t a t i o n . T h e demonstration of the expression of a protein that potentially p r o m o t e s cell a d h e s i o n in the t r o p h o b l a s t m a y well signify t h e true biological f u n c t i o n o f this molecule.
References 1. Herring GM (1972) The organic matrix of bone. In: Bourne GH (ed) The biochemistry and physiology of bone, vol 1. Academic Press, New York, pp 127-189 2. Fisher LW, Whitson SW, Avioli LV, Termine JD (1983) Matrix sialoprotein of developing bone. J Biol Chem 258:12723-12727 3. Fisher LW, Termine JD, Young MF (in press) Human bone sialoprotein: deduced protein sequence and chromosomal localization. J Biol Chem 4. Franzen A, Heinegard D (1985) Isolation and characterization of two sialoproteins present only in bone calcified matrix. Biochem J 232:715-724 5. Fisher L, Hawkins GR, Tuross N, Termine JD (1987) Purification and partial characterization of bone proteoglycans I and II, bone sialoproteins I and II and osteonectin from the mineral compartment of human bone. J Biol Chem 262:9702-9708
6. Kinne RW, Fisher LW (1987) Keratan sulfate proteoglycan in rabbit compact bone is bone sialoprotein II. J Biol Chem 262:10206-10211 7. Oldberg A, Franzen A, Heinegard D (1988) The primary structure of a cell-binding bone sialoprotein. J B iol Chem 263:1943019432 8. Ruoslahti E, Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238:491--497 9. Oldberg A, Franzen A, Heinegard D, Pierschbacher M, Ruoslahti E (1988) Identification of a bone sialoprotein receptor in osteosarcoma cells. J Biol Chem 263:19433-19436 10. Somerman MJ, Fisher LW, Foster RA, Sauk JJ (1988) Human bone sialoproteins I and II enhance fibroblast attachment in vitro. Calcif Tissue Int 43:50-53 11. Mintz KP, Midura RJ, Gehron Robey P, Termine JD, Fisher LW (1990) Attachment properties of non-denatured rat bone sialoprotein. J Bone Miner Res 5:$232 12. Ecarot-Charrier B, Bouchard S, Delloye C (1989) Bone sialoprotein II synthesized by cultured osteoblasts contains tyrosine sulfate. J Biol Chem 264:20049-20053 13. Midura RJ, McQuillan DJ, Benham KJ, Fisher LW, Hascall VC (1990) A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein. J Biol Chem 265:5285-5291 14. Nomura S, Wills AJ, Edwards DR, Heath JK, Hoga BLM (1988) Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization. J Cell Biol 106:44t--450 15. Graham RC, Karnovsky MJ (1966) The early stages of absorption of horseradish peroxidase in the proximal tubules of mouse kidneys: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291-390 16. Cox KH, DeLeon DV, Angerer LM, Angerer RC (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101:485-502 17. Mark MP, Prince CW, Oosawa T, Gay S, Bronckers ALJJ, Butler WT (1987) Immunohistochemical demonstration of a 44kD phosphoprotein in developing rat bones. J Histochem Cytochem 35:707-715 18. Mark MP, Butler WT, Prince CW, Finkelman RD, Ruch JV (1988) Developmental expression of 44-kD bone phosphoprotein (osteopontin) and bone-carboxyglutamic acid (Gla) containing protein (osteocalcin) in calcifying tissues of rat. Differentiation 37:123-136 19. Bianco P, Silverstrini G, Termine JD, Bonucci E (1988) Immunohistochemical localization of osteonectin in human and calf developing bone using monoclonal antibodies. Calcif Tissue Int 43:155-161 20. Bianco P, Fisher LW, Young MF, Kopp JB, Termine JD, Gehron Robey P (1990) The use of synthetic peptide antibodies and in situ hybridization for investigating expression and localization of small proteoglycans of developing bone (biglycan and decorin). In: Cohn DV, Glorieux FH, Martin TJ (eds) Proceedings ASBMR/ICCRH. Elsevier, Amsterdam, pp 201-206 21. Baron R, Neff L, Louvard D, Courtoy JP (1985) Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border. J Cell Biol 101:2210-2222 22: Metsaranta M, Young MF, Sandberg M, Termine JD, Vuorio E (1989) Localization of osteonectin expression in human fetal skeletal tissues by in situ hybridization. Calcif Tissue Int 45:146-152
Calcified Tissue International 9 1991 Springer-Verlag New York Inc.
Molecular and Cellular Biology Expression of Bone Sialoprotein (BSP) in Developing Human Tissues Paolo Bianco, Larry W. Fisher, Marian F. Young, John D. Termine, and Pamela Gehron Robey Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Building 30, Room 106 Bethesda, Maryland 20892, USA Received September 24, 1990, and in revised form December 3, 1990.
Summary. Bone sialoprotein (BSP) and its messenger RNA were localized in developing human skeletal and nonskeletal tissues by means of immunohistochemistry and in situ hybridization. Both protein and mRNA were found in mature, bone-forming cells but not in their immature precursors. In addition, osteoclasts displayed positive immunostaining and high densities of autoradiographic grains by in situ hybridization experiments. BSP was expressed in fetal epiphyseal cartilage cells, particularly in hypertrophic chondrocytes of growth plates. Though neither the protein nor the mRNA were identified in a variety of other connective and nonconnective tissues, an unexpected finding was the expression of BSP in the trophoblast cells of placenta. These findings show that BSP is primarily an osteoblast-derived component of the bone matrix expressed at late stages of differentiation. We have also found that osteoclasts produce BSP, possibly as a mediator of cell attachment to bone. Key words: Bone sialoprotein - - Developing bone - Noncollagenous proteins - - In situ hybridization - - Osteoblast - - Osteoclast - - Cell adhesion.
A sialoprotein of Mr 25,000 was first purified from bovine bone by Herring et al. [I]. Fisher et al. [2] later showed that this 25,000 Mr molecule was most likely a degradation product of a larger -70,000 M r molecule (predicted M r -33,000 by cDNA sequence analysis [3]) and was known briefly as BSP II. Bone sialoprotein (BSP) is found in a variety of mammals, including rat, human, and rabbit [4, 5]. Interestingly, in the rabbit, BSP exists as a keratan sulfate proteoglycan [6]. Both rat [7] and human [3] BSP have recently been cloned and sequenced and have been localized to human chromosome 4 [3]. BSP contains an Arg-Gly-Asp (RGD) sequence, which represents the recognition site for integrins, the cell membrane receptors in a variety of extracellular matrix proteins involved in cell adhesion [8]. In fact, BSP was used to identify an RGD-directed vitronectin-type receptor in rat osteosarcoma cells [9], and mediates fibroblast [10] as well as bone cell [11] attachment in vitro. Evidence that BSP is an endogenous product of normal bone cells comes from in vitro studies with bovine and murine bone-derived cells [2, 12]. Expression in transformed bone Offprint requests to: P. Gehron Robey
cell lines is variable. UMR 106, and in particular UMR-106NIH, produce large amounts of BSP constituitively [13] whereas ROS 17/2.8 produce BSP only after induction [7]. This raises the issue of whether certain subsets (or maturational stages) of bone cells, possibly represented by clonal cell lines, may be specific sites of BSP production in intact tissue. Finally, previous data based on chemical extractions have indicated a highly specific expression of BSP in bone [3, 14], but no immunolocalization studies that document the precise localization or tissue distribution in bone or nonskeletal tissue have been reported to date. In this study we report the expression and localization of BSP in developing human tissues as revealed by immunostaining and in situ hybridization.
Material and Methods Tissues
Human tissues of gestational age 14-17 weeks obtained from therapeutic procedures were placed immediately in Dulbecco's Minimal Essential Medium (DMEM), and dissected within 2 hours. Ribs, calvaria, long bones of the limbs, placentae, and a variety of tissues and viscera (ocular tissue, tendon, skin, skeletal muscle, kidney, sclera, lung) were dissected and fixed for 2 hours or overnight in 4% formaldehyde (freshly made from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.2 and embedded in paraffin. Bone samples were decalcified (0.3 M ethylene diamine tetraacetic acid, 0.15 M NaCI, 0.01 M Tris-HC1 pH 7.2) in buffered EDTA before embedding (2--4 hours), in paraffin (melting point 58-60~ and 5 ~m sections were prepared. Prior to use, slides bearing sections were heated on a warming tray (60~) just until the paraffin melted, and placed in sequential washes of 100% xylene, 100% ethanol, and 70% ethanol (5 minutes in each wash). Antiserum to B S P
A rabbit antiserum (LF 6) raised against purified human BSP was used in this study. Details on the production of the antiserum were given elsewhere [5]. This antibody recognizes only BSP in Western blotting procedures and does not cross-react with osteopontin. Because BAG-75, another bone Sialoprotein isolated from rat bone, has not yet been identified in human bone, it is unknown whether LF-6 cross-reacts with this protein, but it is unlikely based on the immunoblotting procedures and blocking experiments described below. Immunostaining
An indirect immunoperoxidase protocol was used. Deparaffinized sections mounted on Poly-L-lysine-coated slides were exposed to
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P. Bianco et al.: Bone BSP in Developing Tissues
Fig. 2. Immunolocalization of BSP in osteoclasts. (A) An osteoclast adherent to the tip of a primary trabecula. Arrows point to a bandlike zone of immunostaining at the cell/bone interface. (B) A tangentially cut osteoclast, viewed "en face," shows multiple cytoplasmic loci of labeling. Arrows point to "rings" of reaction product around individual nuclei. Counterstain with hemalum. Bar = 20 ~m.
Preparation of RNA Probe
Fig. 1. Immunolocalization of BSP in bone-forming cells. Subperiosteal bone formation in human fetal rib. (A) Labeling of individual osteoid-forming osteoblasts (arrows). Note the presence of a single roundish paranuclear dot of staining (C, arrow). (B) Positive staining of young osteocytes embedded in newly formed bone matrix. Bar = 25 ~m (A), 10 p.m (B), 6 ~m (C). The pattern of cytoplasmic staining in an individual osteoblast is shown in C.
The template for transcription of 35S-labeled RNA probes was a pBluescript plasmid (B6-5g) containing a 1165 base pair insert with the complete protein-encoding sequence of human BSP [3]. Both antisense and sense (control) 35S-UTP-labeled probes (specificity activity 2 x 108 cpm/p~g) were synthesized using the Riboprobe Gemini System (Promega). To synthesize the antisense probe, the plasmid was linearized with BamHI and transcribed using T7 RNA polymerase. To synthesize the sense (control) probe, the plasmid was linearized with Kpn I and transcribed with T3 RNA polymerase. After removal of the template with DNAse RQ 1, the probes were purified by centrifugation on a Sephadex G-50 'spin' column (Pharmacia), and reduced in size by limited alkaline hydrolysis [16].
In Situ
0.3% H202 to inhibit endogenous peroxidase, washed, and exposed to normal goat serum (1:5 in phosphate-buffered saline, PBS, 0.02 M phosphate 0.15 M sodium chloride pH 7.4) for 20 minutes. The antibody to BSP was used at a dilution of 1:40 in PBS, 0.1% bovine serum albumin (BSA). Incubation lasted for 2 hours at room temperature. An affinity purified, peroxidase-labeled goat anti-rabbit IgG antibody was used as the second antibody at a dilution of 1:30 (with an incubation time of 45' at room temperature). Peroxidase activity was revealed using the diamino benzidine reaction [15]. Controls included substitution of normal rabbit serum for the primary antibody, and an antigen absorption test. Blocking experiments were carried out by incubating 1 nmol of purified human BSP with 100 ~1 of the working dilution of the BSP antibody overnight at 4'C, followed by centrifugation at 10,000 x g and subsequent use in immunostaining. The BSP antibody was also preincubated with purified human bone osteopontin (BSP I) to show specificity for BSP and the lack of cross-reactivity in the BSP antiserum with human bone osteopontin.
Chondroitinase Digestion In some experiments, sections were digested with Chondroitin ABC lyase (protease-free, from Proteus Vulgaris, ICN Biochemicals) before immunostaining. The enzyme was used at a concentration of 1.25 U/ml in 0.1 M Tris, 0.05 M calcium acetate, 0.1% BSA, pH 7.2, and incubation lasted for 10 minutes at 37~
Hybridization
Deparaffinized sections were treated with 0.2 N HCI, digested with proteinase K (from Tritirachium album, Sigma), 1 ixg/ml in 10 mM Tris, 2 mM CaC12 for 15 minutes at 37~ washed in PBS, and acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0. The sections were washed in PBS, then in 2 x SSC, dehydrated in ascending concentrations of ethanol, and air dried. The hybridization mixture consisted of 50% formamide, 0.3 M NaC1, 20 mM Tris HC1, pH 8.0, 1 mM EDTA, 1 • Denhardt's solution, tRNA 500 ixg/ml, 10 mM DTT, 10% dextran sulfate, and 2 • 105 cpm/ixl of probe. The volume of hybridization solution added was varied according to the dimensions of the section, and the sections were covered with small plastic squares cut out of heat-sealable, Seal-a-Meal TM pouches. The slides were hybridized overnight at 52~ in a humid atmosphere. The next day, the plastic squares were removed by immersing the slides in 50% formamide, 0.3 M NaC1, 20 mM Tris HC1, pH 8.0, 1 mM EDTA, 1 • Denhardt's, 10 mM DTT. The slides were then sequentially washed in 50% formamide, 4 • SSC, 10 mM DTT at 52~ for 30 minutes, 50% formamide, 2 x SSC, 10 mM DTT at 52~ for 30 minutes; 2 x SSC (4 • 5 minutes) at room temperature. Adventitiously bound probe was removed by digesting the sections with 20 ~g/ml RNase A and 1 i~g/ml RNase T1 in 0.5 M NaC1, 10 mM Tris HC1, pH 8.0, 1 mM EDTA, for 30 minutes at 37~ The slides were then washed in RNase buffer, then in 2 • SSC, 10 mM DTT for 30 minutes at room temperature; 0.1 • SSC, 10 mM DTT at 52~ for 15 minutes; 2 • SSC for 10 minutes at room temperature; then dehydrated and air dried. For autoradiography, the slides were dipped in a 1:1 dilution of Kodak NTB-2 emulsion, allowed to rest vertically at room temperature for 5 hours, then exposed at 4~ for 3-8 days in the presence of desiccant. Exposed
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Fig. 3. Immunolocalization of BSP in placenta. Overview (A) and detail of placental villi (B). Arrows point to areas of surface labeling of the villar profile. Hemalum counterstain. Bar = 40 I~m (A), 25 ixm (B).
slides were developed in Kodak D-19 developer for 2.5 minutes at 15~ stopped in 1% acetic acid, fixed in Kodak fixer, washed in running tap water, counterstained with hemalum, dehydrated in ethanol, cleared in xylene, and mounted in Permount. The slides were viewed in both brightfield and darkfield microscopy.
Results
Immunolocalization of BSP In developing bone, BSP was specifically localized to boneforming cells, i.e., mature osteoblasts, and young osteocytes (osteoid osteocytes) recently entombed in deposited bone matrix (Fig. 1A, B). The staining pattern displayed by bone cells was distinctive in that it consisted of a discrete, sharp, and roundish perinuclear dot, the size, location, and shape of which were consistent with staining of the Golgi apparatus (Fig. 1C). Not all osteoblasts on bone surfaces were positively stained and could reflect different biosynthetic activities of certain cells, or the random orientation of the plane of sectioning which would not necessarily run through the presumed Golgi apparatus of all cells. Preosteogenic ceils in the inner layer of the periostium remained unstained, as did the fibroblastic cells in the outer layer. Osteoclasts also displayed positive immunostaining (Fig. 2). The reaction product in these cells was distributed at the cell/bone interface, and/or as multiple perinculear rings and cytoplasmic dots. Staining of bone matrix was intense but uneven, with focal linear features occasionally reminiscent of cement lines. Focal granular staining was seen in osteoid at the borderline with mature mineralized matrix. Hypertrophic chondrocytes in the growth plate, especially those located at their lateral aspects (i.e., facing the bony collar), consistently showed positive cytoplasmic staining. Occasional cytoplasmic staining of randomly distributed resting chondrocytes was also observed in epiphyseal cartilage. Cartilage matrix remained completely unstained in undigested sections, but was faintly positive in sections predigested with Chondroitin ABC lyase. None of the other connective and nonconnective tissues investigated (ocular tissues, skin, tendon, muscle, kidney etc.) showed any significant staining for BSP. A notable exception was represented by placental villi, where immuno-
staining was seen associated with the surface of ceils of both the syncytial trophoblast and cytotrophoblast (Fig. 3). Foci of " s p o t t y " placental calcification and the endothelial lining of occasional capillaries in the core of villi were also stained. No staining was seen in control sections incubated with either normal rabbit serum of BSP-absorbed antiserum. The immunostaining was completely blocked by preincubation of the antibody with highly purified human bone sialoprotein indicating the specificity of the antibody and the lack of cross-reactivity with other proteins such as osteopontin and the potential human analog of BAG-75. In addition, positive staining was not affected by absorption of the antiserum with osteopontin.
Localization of BSP mRNA In developing subperiosteal bone, BSP mRNA was localized to mature osteoblasts on bone surfaces and young osteocytes within bone matrix (Fig. 4A-D), but not in the preosteogenic and fibrous layers of the periosteum. Autoradiographic grains were observed over Howship's lacunae (Fig. 4E-F), which suggested that osteoclasts were also labeled. The morphology of the labeled cells at those sites was somewhat obscured by the high density of the grains themselves. Individual osteoclasts that had become attached from bone (most likely by the sectioning procedure) were easier to identify in our sections on the basis of their size and multinuclearity. Such cells also displayed high hybridization signal (Fig. 4G, H). In nonbone tissues, positive hybridization signals were detected only in epiphyseal cartilage and placenta. In epiphyseal cartilage, relatively low levels of BSP m R N A (as judged by grain densities) were observed in resting and proliferating chondrocytes, and higher levels in hypertrophic chondrocytes, indicating a gradient of increasing expression of BSP mRNA in the growth plate (Fig. 5A, B). In placenta, cells of cyto- and syncytial trophoblast at the surface of villi displayed strong hybridization signals (Fig. 6). No positive labeling was seen over any of the other tissues investigated (skin, kidney, eye, muscle, etc.) and in sections hybridized with the sense probe. The distribution of BSP in matrix and cells, along with localization of BSP mRNA, is summarized in Table 1.
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Fig. 4. Localization of BSP mRNA in bone. Brightfield and darkfield images of the bony collar of human developing femur at low power (A, B, bar = 50 ~m) and high power (C, D). (Bar = 20 Ixm). Osteoblasts at the bone surface (large arrows) and osteocytes within the matrix (small arrows) are labeled. Bar = 50 0.m. Brightfield and darkfield images of trabeculae of primary spongiosa (E, F). Note the concentration of autoradiographic grains over a Howship's lacuna (arrow). Bar = 50 gin. Detail of a labeled osteoclast lying in proximity of a bone trabecula (G, It). Nomarski optics is used in (G) to give enough contrast to the bone trabecula (bt). Autoradiographic grains are shown in (H), where arrows point to individual nuclei. Bar = 30 p~m.
Discussion
The present study conclusively demonstrates that in developing human bone, osteoblasts and young osteocytes are the primary source of bone sialoprotein. The peculiar intracellular staining pattern displayed by bone-forming cells with our anti-BSP antibody is strongly reminiscent of the one described by Mark et al. [17] for osteopontin in rat bone cells, and shown to result from localization of osteopontin in the Golgi apparatus [18]. This would be consistent with the known occurrence of siaiylation in the Golgi and the high content of sialic acid of both proteins, which would tend to cause accumulation in this organelle. Interestingly, not all osteoblasts and young osteocytes were stained, the ramifications of which are not clear, but could represent either subpopulations of cells or differences in the plane of section. It is of interest that only mature bone-forming cells (which include osteoblasts sitting at bone surfaces and young
osteocytes) express BSP detectable by these methods. The level o f BSP expression was undetectable in less mature cells, such as preosteoblasts in the inner periostium, compared with the more differentiated cells. This " l a t e " expression in bone-forming cells is different from that of other noncollagenous components of bone matrix, such as osteonectin [19] and the small proteoglycans biglycan (PG I) and decorin (PG II) which are expressed earlier in the differentiation pathway of osteoblasts [20], but similar to that of osteopontin [17] with the exception that small mononuclear cells in the marrow were osteopontin positive, but not BSP positive at this early stage of development. The programmed expression of specific matrix components by bone-forming cells at different maturational stages raises the possibility that distinct biosynthetic profiles mark specific phenotypes within the bone cell lineage. An intriguing finding of this study is the localization of BSP protein and of the corresponding m R N A in osteoclasts.
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Fig. 5. Detection of BSP mRNA in epiphyseal cartilage. Note the higher grain density over hypertrophic chondrocytes (bottom) as compared with proliferating cells (top). Brightfield (A) and darkfield (B) images. Bar = 50 ~m.
Fig. 6. Detection of BSP mRNA in placenta. Brightfield (A) and darkfield (B) images of a placental villus showing strong hybridization signal over trophoblastic cells (arrows). Bar = 60 txm. Though the localization of the protein in osteoclasts is clear in terms of cell morphology, localization of matrix proteins within osteoclasts may be due to either diffusion artifacts or localization of molecules in the process of being resorbed rather than synthesized. Nevertheless, (1) antibodies to other matrix proteins used on the same materials did not yield any staining of osteoclasts; (2) a discrete cytoplasmic staining pattern, consisting of multiple perinuclear rings, was observed in osteoclasts, which might be consistent with a Golgi staining, given the "ring-like" perinuclear distribution of the Golgi apparatuses in osteoclasts [21]; (3) the observation of positive signals, indicative of mRNA, over resorption lacunae and individual osteoclasts in our in situ hybridization experiments supports the notion that osteoclasts can synthesize BSP. Given the postulated role of the BSP in mediating cell attachment, the possibility that osteoclasts may use endogenously produced cell adhesion molecules to attach to bone surfaces is most interesting. Preliminary results indicate that isolated chicken osteoclasts do indeed adhere to BSP in vitro (F. P. Ross, personal communication).
Our results show that although restricted to a limited number of cell types, BSP expression is not totally unique to bone cells. The expression of BSP mRNA in epiphyseal cartilage predominantly in hypertrophic chondrocytes, and the observed gradient of increasing expression along the direction of chondrocyte maturation in growth plates are reminiscent of similar reported localizations of other bone-enriched noncollagenous proteins and/or of the c o r r e s p o n d i n g mRNAs, such as osteonectin [14, 19, 22] and osteopontin [18]. It is worth noting here that the expression of high levels of BSP mRNA might be an additional example of the appearance or up-regulation of certain biosynthetic or phenotypic features characteristic of mature osteoblasts, including alkaline phosphatase activity, in hypertrophic chondrocytes of growth plates. In addition to the hard tissue localization, trophoblasts are the only nonskeletal source of BSP at 14 weeks of gestation. Thus, for trophoblasts, phenotypic modulations cannot account for what could be called an "ectopic" expression of a bone-enriched protein. The known occurrence of
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Table 1. Distribution of BSP and BSP mRNA in human developing skeletal and nonskeletal tissues
Bone Matrix Preosteoblasts Mature osteoblasts Osteocytes Osteoclasts Cartilage Matrix Chondrocytes Resting Proliferating Hypertrophic Nonskeletal tissues Soft tissues Parenchymal viscera Trophoblast
BSP
BSP mRNA
+ + + +
+ + +
-+a • • +
• +
+
+
BSP = bone sialoprotein; + = positive; - = negative; • = variably positive in some cells a Weakly positive only after ABCase
d y s t r o p h i c m i n e r a l i z a t i o n (as o p p o s e d to m a t r i x - m e d i a t e d m i n e r a l i z a t i o n ) in p l a c e n t a , a n d t h e i m m u n o l o c a l i z a t i o n of B S P in s u c h p l a c e n t a l s p o t t y foci o f m i n e r a l d e p o s i t i o n , also does n o t p r o v i d e a s a t i s f a c t o r y a n a l o g y with skeletal tissues. However, the association of BSP and mineral may not be fortuitous. It is i n t e r e s t i n g to n o t e t h a t b o t h o s t e o c l a s t s a n d t r o p h o b l a s t cells are i n v o l v e d in t h e f o r m a t i o n of s y n c y t i a , for w h i c h cell-cell a d h e s i o n is n e c e s s a r y , a n d t h a t t h e adhesion b e t w e e n fetal a n d m a t e r n a l tissues is o n e o f the m o s t intriguing a n d l e a s t u n d e r s t o o d p h e n o m e n a of g e s t a t i o n . T h e demonstration of the expression of a protein that potentially p r o m o t e s cell a d h e s i o n in the t r o p h o b l a s t m a y well signify t h e true biological f u n c t i o n o f this molecule.
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